MACC1 siRNA lentivirus transduction particles (MACC1 siRNA sequence: forward, 5′-AAGAGGGGACGGGGACACGGCTT-3′ and reverse, 5′-TTGGCGAACCGGAACAGGGGACG-3′) and negative siRNA control were acquired from Sigma-Aldrich. MACC1-knockdown plasmid (PCMV6-AC-GFP) and empty carrier (PCMV6-Entry) were bought from Origene Inc. Colon cancer cells were inoculated into six-hole plates and grew overnight to 80% and then mixed. Subsequently, the cells were transfected with siRNA-MACC1 or PCMV-MACC1 by using AmaxaNucleofector (Amaxa, Kölner) according to the specification of the manufacturer. Downregulation and overexpression efficiency was determined with Western blot (WB). Cell toxicity experiment was performed using CCK-8, and the grouping and drug concentration were the same as those in Section 1.2.
Negative sirna control
Manufactured by Merck Group
Negative siRNA control is a laboratory tool used to establish baseline levels of gene expression in RNA interference (RNAi) experiments. It serves as a reference point to evaluate the specific effects of targeted siRNA molecules on gene silencing. The negative control does not target any known gene and helps determine the background level of gene knockdown in the absence of a specific siRNA.
Automatically generated - may contain errors
2 protocols using negative sirna control
1
Modulation of MACC1 in Colon Cancer
2
Transfection of Cells with siRNA
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In this study, 2 × 105 cells/well in six-well dishes and 2 × 104 cells/well in confocal plates were seeded 24 h before transfection. After 24 h the media was removed and transfection was proceeded with fresh media containing 20 ng/ml TNFα without and with 20 nM scrambled smart pool DNase I siRNA or IL-1β siRNA (Dharmacon) and negative siRNA control (Sigma) using Lipofectamine 2000 (Invitrogen). The cells were harvested after 48 h after transfection. siRNA against P62 was used as a positive control for transfection in separate wells.
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